• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.392-400
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• 2019

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ${\Delta}L-CgCAU$ in a 5-L fed-batch fermentation with a single distribution $M_w$ of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1583-1591
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• 2020

CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.

• Journal of Microbiology
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• v.34 no.4
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• pp.355-362
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• 1996

Complementation cloning of the argC, E, B, D, F, and G genes in Corynebacterium glutamicum was done by transforming the genomic DNA library into the corresponding arginine auxotrophs fo Escherichia coli. Recombinant plasmids containing 6.7 kb and 4.8kb fragments complementing the E. coli argB mutant were also able to complement the E. coli argC, E, A, D, and F mutants, indicating the clustered organization of the arginine biosynthetic genes within the cloned DNA fragments. The insert DNA fragments in the recombinant plasmids, named pRB1 AND pRB2, were physically mapped with several restriction enzymes. By further subcloning the entire DNA fragment containing the functions and by complementation analysis, we located the arg genes in the order of ACEBDF on the restriction map. We also determined the DNA nucleotide sequence of the fragment and report here the sequence of the argB gene. When compared to that with the mutant strain, higher enzyme activity of N-acetylglutamate kinase was detected in the extract of the mutant carrying the plasmid containing the putative argB gene, indicating that the plasmid contains a functional argB gene. Deduced amino acid sequence of the argB gene shows 45%, 38%, and 25% identity to that from Bacillus strearothermophilus, Bacillus substilus, and E. coli respectively. Our long term goal is genetically engineering C. glutamicum which produces more arginine than a wild type strain does.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.174-179
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• 1991

Protoplast fusion was carried out between Brevibacterium flavum and Corynebacterium glutamicum. For the protoplast fusion, various mutants were isolated from Brevibacterium flavum ATCC 21493 and Corynebacteriurn glutamicum ATCC 21831. The optimum conditions for protoplast fusion of these mutants were examined. In the present work, the authors obtained a fusant, MWF 9031, by the intergeneric protoplast fusion between Brevibacterium flavum 108-125 and Corynebacterium glutamicum 41-214A, which was excellent in L-arginine fermentation. Fusant MWF 9031 was found to accumulate a large amount of L-arginine reached 32.5 mg/ml with a medium containing 10% glucose. The fusant possessed intermediate characteristics between the parental strains and the stability was found to retain for 60 days.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.256-263
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• 1994

The aceB gene, encoding for malate synthase, one of the key enzymes of glyoxylate bypass, was isolated from a pMT1-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli aceB mutant on an acetate minimal medium. The aceB gene was closely linked to aceA, separated by 598 base pairs, and transcribed in divergent direction. The aceB expressed a protein product of Mr 83, 000 in Corynebacterium glutamicum which was unusually large compared with those of other malate synthases. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 2, 217 base pairs which encodes a protein with the molecular weight of 82, 311 comprising 739 aminoo acids. The putative protein product showed only limited amino acid-sequence homology to its counteliparts in other organisms. The N-terminal region of the protein, which shows no apparent homology with the known sequences of other malate synthases, appeared to be responsible for the protein s unusually large size. A potential calciumbinding domain of EF-hand structure found among eukaryotes was detected in the N-terminal region of the deduced protein.

• Journal of the East Asian Society of Dietary Life
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• v.5 no.1
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• pp.93-99
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• 1995

For the improvement of the L-lysine productivity of Brevibacterium flavum and Corynebacterium glutamicum, fusants were induced by interspecific protoplast fusion of Bacillus subtilis with C. glutamicum and B. flavum. The following results were obtained through protoplast formation of strains condition of protoplast fusion, characteristics of the fusants, and the productivity of lysine form starch. B. flavum BF-5 and C. glutamicum protoplasts were made by the treatment of 0.3unit/$m\ell$ of penicillin G at the early stationary growth phase for 2 hours followed by incubation with 10mg/$m\ell$ of lysozyme at 37$^{\circ}C$ for 120 min. When a mixture of the protoplast was treated with 30% PEG(M.W.6,000) solution containing 50mM CaCl2 at optimal conditions, the intergeneric fusion frequency between protoplasts of C. glutamicum CG-2 and B. subtilis BD 224 was 7.1${\times}$105. The genetic properties on the L-lysine producing fusants were compared with those of parental strains. As a results, the intergeneric fusants were completed in each auxotrophic requirement, resistances for S-(2-amino-ethyl)-L-cysteine and kanamycine were confirmed. And one of fusants selected, FBB-41 were found to be genetically stable fusants. The aspartokinase activity of FBB-41 strain increased than that of the parent strain.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.336-339
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• 2002

A total of 100 Corynebacterial clones exerting a regulatory effect on the aceB promoter of Corynebacterium glutamicum were isolated by utilizing a reporter carrying the enteric lacZ gene fused to the promoter. The isolated clones were classified into 3 groups of A, B, and C, according to their color of colonies. Escherichia coli cells carrying clones in groups A and B showed a $90\%\;and\;50\%$ reduction in ${\beta}$-galactosidase activity, respectively. The introduction of group A clones into C. glutamicum also resulted in an almost complete reduction in the expression of the aceA and aceB genes, suggesting that the clones express repressor-like proteins for the genes. Although white colonies were formed on plates containing X-gal, E. coli cells carrying one of the clones in group C exhibited intact ${\beta}$-galactosidase activity. The result suggests that the clone may encode proteins that prevent the cells from accumulating the chromogenic compound, X-gal.

• Korean Journal of Organic Agriculture
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• v.7 no.1
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• pp.105-113
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• 1998

This study was conducted to clarify the effects of various rhizobacteria in the root zone in terms of Azospirillum sp., Rhodopseudomonas sp., Pseudomonas sp., fusant of Bacillus sp. and Corynebacterium glutamicum on the growth of hydroponically grown cucumber plants. Densities in bacterial cells of fusant of Bacillus sp. and Corynebacterium glutamicum at different substrates were in the order of cocopeat > rockwool > nutrient solutions at 4 days after bacterialization. Plant growth promoting effects of the various rhizobacteria on the growth of hydroponically grown cucumber plants were in the order of Azospirillum sp. > Rhodopseudomonas sp. $\ge$ fusant of Bacillus sp. and Corynebacterium glutamicum > Pseudomonas sp. > control.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1370-1375
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• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.1010-1017
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• 2007

Two alternative pathways for methionine biosynthesis are known in Corynebacterium glutamicum: one involving transsulfuration (mediated by metB and metC) and the other involving direct sulfhydrylation (mediated by metY). In this study, MetB (cystathionine ${\gamma}-synthase$) and MetY (O-acetylhomoserine sulfhydrylase) from C. glutamicum were purified to homogeneity and the biochemical parameters were compared to assess the functional and evolutionary importance of each pathway. The molecular masses of the native MetB and MetY proteins were measured to be approximately 170 and 280 kDa, respectively, showing that MetB was a homotetramer of 40-kDa subunits and MetY was a homohexamer of 45-kDa subunits. The $K_m$ values for the O-acetylhomoserine catalysis effected by MetB and MetY were 3.9 and 6.4 mM, and the maximum catalysis rates were $7.4\;(k_{cat}=21\;S^{-1})\;and\;6.0\;(k_{cat}=28\;S^{-1})\;{\mu}mol\;mg^{-1}\;min^{-1}$, respectively. This suggests that both MetB and MetY can be comparably active in vivo. Nevertheless, the $K_m$ value for sulfide ions by MetY was 8.6mM, which was too high, considering the physiological condition. Moreover, MetB was active at a broad range of temperatures $(30\;and\;65^{\circ}C)$ and pH (6.5 and 10.0), as compared with MetY, which was active in a range from 30 to $45^{\circ}C$ and at pH values from 7.0 to 8.5. In addition, MetY was inhibited by methionine, but MetB was not. These biochemical data may provide insight on the role of the parallel pathways of methionine biosynthesis in C. glutamicum with regard to cell physiology and evolution.